The present invention relates to media comprising purified antimicrobial peptides, pore forming agents, and/or cell surface receptor binding compounds and their use for the storage and preservation of organs prior to transplant.
A wide variety of organs, including kidneys, lungs, livers, hearts, pancreases, and small intestines are routinely and successfully transplanted. These organs are obtained either from living donors or from cadaveric sources.
In 1998, a total of 12,166 kidney transplants were performed in the United States by programs tracked by the UNOS Transplant Patient DataSource. A total of 45,189 people were on the waiting lists for kidneys as of Sep. 30, 1999. Over 20,000 kidneys were transplanted between Jul. 1, 1995 and Jun. 30, 1997. The graft survival rate for these 2(transplanted kidneys was 93.4% after three months.
The ability to store organs for two or three days prior to transplantation allows sufficient time for histo-compatibility testing of donor and recipient, transport of the organ between transplant centers, preoperative preparation of the recipient, preliminary donor culture testing, and vascular repair of the organ if needed. The efficacy of organ transplantation depends in part on how well the organ is preserved prior to transplantation. Two methods are used to preserve organs prior to transplant: hypothermic storage and continuous pulsatile perfusion. Hypothermic storage by simple cold storage methods involves removal of an organ from a donor followed by rapid cooling. Cooling is achieved by a combination of external cooling and a short period of perfusion with a chilled medium to reduce the core temperature of the organ as quickly as possible. The organs are then immersed in a flush-out medium at from 0xc2x0 C. to 4xc2x0 C. Continuous pulsatile perfusion involves the continuous infusion of organs with a preservation solution designed to prevent low temperature injury.
A number of media have been developed for infusing and preserving organs prior to transplantation. Examples of such media include VIASPAN (also known as University of Wisconsin solution; Barr Laboratories, Pomona, N.Y.), University of Wisconsin Machine Perfusion Solution, Hypertonic Citrate Solution, HTK Solution, HTK Solution of Bretschneider, Phosphate Buffered Sucrose, EuroCollins Solution, and Collins C2 Solution. However, none of these media are able to extend the preservation of organs past about 72 hours using cold storage methods. Additional preservation time would be useful for tests and for transportation of the organs. Furthermore, media that increase preservation time also can be expected to provide healthier organs for transplants performed within 72 hours.
Accordingly, what is needed in the art are improved media for preserving and storing organs prior to transplant. Such media should be able to extend the preservation period past 72 hours and provide organs with increased functionality upon transplant.
The present invention relates to media comprising antimicrobial polypeptides or pore forming agents and/or cell surface receptor binding compounds and their use for the storage and preservation of organs prior to transplant.
The present invention is not limited to any particular media or formulation. Indeed, a variety of medias and formulations are contemplated. In some embodiments, the present invention provides compositions comprising a purified antimicrobial polypeptide and hydroxyethyl starch. The present invention is not limited to any particular antimicrobial peptide. Indeed a variety of antimicrobial peptides are contemplated, including, but not limited to, those identified by SEQ ID NOs:1-96. In some preferred embodiments, the antimicrobial peptide is a defensin. The present invention is not limited to any particular defensin. Indeed, the use of a variety of defensins is contemplated, including, but not limited to those identified by SEQ ID NOs:37-96. In particularly preferred embodiments, the antimicrobial peptide is bovine dodecapeptide or BNP-1 (SEQ ID NO: 37). In some preferred embodiments, the antimicrobial polypeptide or defensin comprises D-amino acids. In some embodiments, the antimicrobial peptide and hydroxyethyl starch are in solution. The media of the present invention are not limited to any particular concentration of antimicrobial peptide. Indeed, a range of concentrations are contemplated (e.g., from about 0.01 to 1000 mg/l and preferably from about 0.1 to 5 mg/1). The present invention is not limited to any particular concentration of hydroxyethyl starch. Indeed, a range of concentrations are contemplated (e.g., from about 1 to 200 g/l). In some embodiments, the media further comprises a cell surface receptor binding compound. The present invention is not limited to any particular cell surface receptor binding compound. Indeed, a variety of cell surface receptor binding compounds are contemplated, including, but not limited to IGF-1, EGF, NGF, and substance P.
In other embodiments, the present invention provides compositions comprising an antimicrobial polypeptide and an impermeant anion selected from the group consisting of lactobionic acid and gluconate. In some preferred embodiments, the antimicrobial polypeptide and the impermeant ion are in solution. The present invention is not limited to any particular antimicrobial peptide. Indeed a variety of antimicrobial peptides are contemplated, including, but not limited to, those identified by SEQ ID NOs:1-96. In some preferred embodiments, the antimicrobial peptide is a defensin. The present invention is not limited to any particular defensin. Indeed, the use of a variety of defensins is contemplated, including, but not limited to those identified by SEQ ID NOs:37-96. In some preferred embodiments, the antimicrobial polypeptide or defensin comprises D-amino acids. In particularly preferred embodiments, the antimicrobial peptide is bovine dodecapeptide or BNP-1 (SEQ ID NO: 37). The media of the present invention are not limited to any particular concentration of antimicrobial peptide. Indeed, a range of concentrations are contemplated (e.g., from about 0.01 to 1000 mg/l and preferably from about 0.1 to 5 mg/l). The media of the present invention are not limited to any particular concentration of impermeant ion. Indeed, a range of concentrations are contemplated (e.g., from about 1 to 500 mM). In some embodiments, the media further comprises a cell surface receptor binding compound. The present invention is not limited to any particular cell surface receptor binding compound. Indeed, a variety of cell surface receptor binding compounds are contemplated, including, but not limited to IGF-1, EGF, NGF, and substance P. In some preferred embodiments, the media does not require the use of hydroxyethyl starch.
In other embodiments, the present invention provides compositions comprising an antimicrobial polypeptide and glutathione. In some preferred embodiments, the antimicrobial polypeptide and the impermeant ion are in solution. The present invention is not limited to any particular antimicrobial peptide. Indeed a variety of antimicrobial peptides are contemplated, including, but not limited to, those identified by SEQ ID NOs:1-96. In some preferred embodiments, the antimicrobial peptide is a defensin. The present invention is not limited to any particular defensin. Indeed, the use of a variety of defensins is contemplated, including, but not limited to those identified by SEQ ID NOs:37-96. In some preferred embodiments, the antimicrobial polypeptide or defensin comprises D-amino acids. In particularly preferred embodiments, the antimicrobial peptide is bovine dodecapeptide or BNP-1 (SEQ ID NO: 37). The media of the present invention are not limited to any particular concentration of antimicrobial peptide. Indeed, a range of concentrations are contemplated (e.g., from about 0.01 to 1000 mg/l and preferably from about 0.1 to 5 mg/l). The media of the present invention are not limited to any particular concentration of glutathione. Indeed, a range of concentrations are contemplated (e.g., from about 0.1 to 100 mM). In some embodiments, the media further comprises a cell surface receptor binding compound. The present invention is not limited to any particular cell surface receptor binding compound. Indeed, a variety of cell surface receptor binding compounds are contemplated, including, but not limited to IGF-1, EGF, NGF, and substance P. In some preferred embodiments, the media does not require the use of hydroxyethyl starch.
In further embodiments, the present invention provides compositions comprising a purified antimicrobial polypeptide and an ex vivo internal organ. The present invention is not limited to any particular internal organ. Indeed, a variety of internal organs are contemplated, including, but not limited to kidneys, hearts, lungs, small intestines, large intestines, livers, and pancreases. The present invention is not limited to organs from any particular species of animal. Indeed, use of organs from a variety of animals is contemplated, including organs from humans, pigs, and dogs. The present invention is not limited to any particular antimicrobial peptide. Indeed a variety of antimicrobial peptides are contemplated, including, but not limited to, those identified by SEQ ID NOs:1-96. In some preferred embodiments, the antimicrobial peptide is a defensin. The present invention is not limited to any particular defensin. Indeed, the use of a variety of defensins is contemplated, including, but not limited to those identified by SEQ ID NOs:37-96. In particularly preferred embodiments, the antimicrobial peptide is bovine dodecapeptide or BNP-1 (SEQ ID NO: 37). In some preferred embodiments, the antimicrobial polypeptide or defensin comprises D-amino acids. The media of the present invention are not limited to any particular concentration of antimicrobial peptide. Indeed, a range of concentrations are contemplated (e.g., from about 0.01 to 1000 mg/l and preferably from about 0.1 to 5 mg/l). In some embodiments, the compositions further comprise a macromolecular oncotic agent. The present invention is not limited to any particular macromolecular oncotic agent. Indeed, a variety of macromolecular oncotic agents are contemplated, including, but not limited to hydroxyethyl starch, dextran, and glucose. In other embodiments, the composition further comprises an impermeant anion. The present invention is not limited to any particular impermeant anion. Indeed, a variety of impermeant anions are contemplated, including, but not limited to, gluconate and lactobionic acid. In still further embodiments, the compositions comprise glutathione. In some embodiments, the compositions further comprise a cell surface receptor binding compound. The present invention is not limited to any particular cell surface receptor binding compound. Indeed, a variety of cell surface receptor binding compounds are contemplated, including, but not limited to IGF-1, EGF, NGF, and substance P. In some preferred embodiments, the media does not require the use of hydroxyethyl starch.
In still other embodiments, the present invention provides methods comprising a) providing cellular material and a solution comprising a purified antimicrobial polypeptide and b) storing the cellular material in said solution comprising a purified antimicrobial peptide. The present invention is not limited to the storage of any particular cellular material. Indeed, a variety of cellular materials are contemplated, including but not limited to internal organs, skin, and gametes. In some preferred embodiments, the cellular material is an internal organ. The present invention is not limited to any particular internal organ. Indeed, a variety of internal organs are contemplated, including, but not limited to kidneys, hearts, lungs, small intestines, large intestines, livers, and pancreases. The present invention is not limited to organs from any particular species of animal. Indeed, use of organs from a variety of animals is contemplated, including organs from humans, pigs, and dogs. In some embodiments, the internal organ is infused with the solution. The present invention is not limited to any particular antimicrobial peptide. Indeed a variety of antimicrobial peptides are contemplated, including, but not limited to, those identified by SEQ ID NOs:1-96. In some preferred embodiments, the antimicrobial peptide is a defensin. The present invention is not limited to any particular defensin. Indeed, the use of a variety of defensins is contemplated, including, but not limited to those identified by SEQ ID NOs:37-96. In particularly preferred embodiments, the antimicrobial peptide is bovine dodecapeptide or BNP-1 (SEQ ID NO: 37). In some preferred embodiments, the antimicrobial polypeptide or defensin comprises D-amino acids. The media of the present invention are not limited to any particular concentration of antimicrobial peptide. Indeed, a range of concentrations are contemplated (e.g., from about 0.01 to 1000 mg/l and preferably from about 0.1 to 5 mg/l). In some embodiments, the compositions further comprise a macromolecular oncotic agent. The present invention is not limited to any particular macromolecular oncotic agent. Indeed, a variety of macromolecular oncotic agents are contemplated, including, but not limited to hydroxyethyl starch, dextran, and glucose. In other embodiments, the composition further comprises an impermeant anion. The present invention is not limited to any particular impermeant anion. Indeed, a variety of impermeant anions are contemplated, including, but not limited to, gluconate and lactobionic acid. In still further embodiments, the compositions comprise glutathione. In some embodiments, the compositions further comprise a cell surface receptor binding compound. The present invention is not limited to any particular cell surface receptor binding compound. Indeed, a variety of cell surface receptor binding compounds are contemplated, including, but not limited to IGF-1, EGF, NGF, and substance P. In some preferred embodiments, the media does not require the use of hydroxyethyl starch.
In still further embodiments, the present invention provides compositions comprising a cell surface receptor binding compound and hydroxyethyl starch. The present invention is not limited to any particular cell surface receptor binding compound. Indeed, a variety of cell surface receptor binding compounds are contemplated, including, but not limited to IGF-1,EGF, NGF, and substance P.
In other embodiments, the present invention provides compositions comprising a cell surface receptor binding compound and an internal organ. In some embodiments, the compositions further comprise a macromolecular oncotic agent. The present invention is not limited to any particular macromolecular oncotic agent. Indeed, a variety of macromolecular oncotic agents are contemplated, including, but not limited to hydroxyethyl starch, dextran, and glucose. In other embodiments, the composition further comprises an impermeant anion. The present invention is not limited to any particular impermeant anion. Indeed, a variety of impermeant anions are contemplated, including, but not limited to, gluconate and lactobionic acid. In still further embodiments, the compositions comprise glutathione. In some preferred embodiments, the media does not require the use of hydroxyethyl starch.
In some embodiments, the present invention provides compositions comprising trehalose and hydroxyethyl starch. In some preferred embodiments, the trehalose and hydroxyethyl starch are in solution. The present invention is not limited to any particular concentration of trehalose. Indeed, a range of concentrations are contemplated (e.g., from about 1 mM to 30 mM). In some embodiments, the compositions further comprise an antimicrobial peptide and/or cell surface receptor binding compound. In some embodiments, the compositions further comprise a cell surface receptor binding compound. The present invention is not limited to any particular cell surface receptor binding compound. Indeed, a variety of cell surface receptor binding compounds are contemplated, including, but not limited to IGF-1, EGF, NGF, and substance P. The present invention is not limited to any particular antimicrobial peptide. Indeed a variety of antimicrobial peptides are contemplated, including, but not limited to, those identified by SEQ ID NOs:1-96. In some preferred embodiments, the antimicrobial peptide is a defensin. The present invention is not limited to any particular defensin. Indeed, the use of a variety of defensins is contemplated, including, but not limited to those identified by SEQ ID NOs:37-96. In particularly preferred embodiments, the antimicrobial peptide is bovine dodecapeptide or BNP-1 (SEQ ID NO: 37). The media of the present invention are not limited to any particular concentration of antimicrobial peptide. Indeed, a range of concentrations are contemplated (e.g., from about 0.01 to 1000 mg/l and preferably from about 0.1 to 5 mg/l). In some embodiments, the compositions further comprise a macromolecular oncotic agent. The present invention is not limited to any particular macromolecular oncotic agent. Indeed, a variety of macromolecular oncotic agents are contemplated, including, but not limited to hydroxyethyl starch, dextran, and glucose. In other embodiments, the composition further comprises an impermeant anion. The present invention is not limited to any particular impermeant anion. Indeed, a variety of impermeant anions are contemplated, including, but not limited to, gluconate and lactobionic acid. In still further embodiments, the compositions comprise glutathione.
In other embodiments, the present invention provides a kit comprising a vessel containing a solution comprising a compound selected from the group consisting of lactobionate and hydroxyethyl starch; and a vessel containing an antimicrobial polypeptide. In some embodiments, the antimicrobial polypeptide is BNP-1. In other embodiments, the vessel containing an antimicrobial polypeptide further comprises a cell surface receptor binding compound. In further embodiments, the cell surface receptor binding compound is selected from the group consisting of IGF-1, EGF, NGF, and substance P. In some embodiments, the kit further comprises instructions for combining said solution and the antimicrobial polypeptide.
In still further embodiments, the present invention provides processes for producing a storage solution comprising providing a solution comprising a compound selected from the group consisting of hydroxyethyl starch and lactobionate and a purified antimicrobial polypeptide; and combining said solution with the purified antimicrobial polypeptide. In some embodiments, the method further comprising the steps of providing at least one cell surface receptor binding compound and combining the at least one cell surface receptor binding compound with the solution and the antimicrobial polypeptide.
In some preferred embodiments, the present invention provides a composition comprising hydroxyethyl starch or lactobionate and an antimicrobial polypeptide for use as an organ storage or perfusion solution. In some embodiments, the composition further comprising a cell surface receptor binding compound. In other preferred embodiments, the present invention provides a composition comprising a purified antimicrobial polypeptide (e.g., BNP-1) and at least one purified cell surface receptor binding compound (e.g., IGF-1, EGF, NGF, and substance P), for use as a supplement for organ storage solutions.
In some embodiments, the media described herein further comprise a microtubule stabilizing agent selected from the group consisting of taxol, discodermolide, epothilone A and B, vinblastine, and vinchristine.
In still further embodiments, the present invention provides methods and compositions for stabilizing microtubules in cells, tissues, or organs, either in vitro, in vivo, or ex vivo. In preferred embodiments, the compositions comprise a defensin (e.g., BNP-1). In other preferred embodiments, the compositions comprise a cell surface receptor binding compound, impermeant anion, energy source, or macromolecular oncotic agent as described in more detail above. In other particularly preferred embodiments, the present invention provides a composition comprising a defensin (e.g., BNP-1) for use in stabilizing microtubules and/or actin filaments. In still other embodiments, the present invention provides methods and processes comprising providing a cell, tissue or organ, and a composition comprising a purified defensin, and treating the cell, tissue, or organ under conditions such that the cytoskeleton of the cell tissue, or organ is stabilized. In particularly preferred embodiments, microtubules and and/or actin filaments are stabilized. In still other particularly preferred embodiments, the defensin id BNP-1 (SEQ ID NO: 37).
In still further embodiments, the present invention provides a composition substantially as described in any of the examples herein.